Diarrhea has long been recognized as an adverse result of antibiotic therapy in humans. The severity of the diarrhea ranges from a mild, self-limiting process to a fulminating, life- threatening pseudomembranous colitis (PMC). Toxigenic Clostridium difficile located in the intestinal tract is known to be a major cause of antimicrobial associate colitis, and it is likely the sole cause of PMC in humans. Clostridium difficile is also an etiological agent in certain intestinal diseases not related to antimicrobial therapy. The pathogenic mechanism(s) by which Clostridium difficile brings about these intestinal diseases remains poorly understood. However, it is thought to be dependent, in part, upon two biochemically and immunochemically distinct toxins toxin A, an enterotoxin and toxin S, a cytotoxin. Relatively little detail is known of the biochemical, immunochemical, genetic and physiological properties of these toxins. Therefore, the purpose of this proposal is to obtain a better understanding of the structure and function of these two toxins. This will be achieved with a more rigorous biochemical, immunochemical, genetic and physiological characterization of these toxins. First, monoclonal antibodies specific for each toxin will be produced and characterized and these will be used to more rigorously defile biochemical parameters of the toxins. Next, monoclonal antibodies will be used to isolate toxin gene fragments and the genes will be cloned and characterized, helping to define further their biochemical and genetic relationships. Then toxin deficient bacteria will be produced through chemical mutagenesis, and these will be used to study altered toxins they might produce. Finally, in vitro mutagenesis of cloned toxin genes will be used to produce altered toxins in E. coli and their toxigenic function will be studied.